The ability to simultaneously acquire objective physiological measures of emotion concurrent with fMRI holds the promise to enhance our understanding of the biological bases of affect, and thus improve our knowledge of the neural circuitry underlying psychiatric disorders. However, the vast majority of neuroimaging studies to date examining emotion have not anchored the examination of emotion-responding circuitry to objective measures of emotional processing. To that end, we acquired electromyographic (EMG) activity of a valence-sensitive facial muscle involved in the frowning response (corrugator muscle) concurrent with fMRI while twenty-six human participants viewed negative and neutral images. Trial-by-trial increases in corrugator EMG activity to negative pictures were associated with greater amygdala activity, and a concurrent decrease in ventromedial prefrontal cortex activity. Thus, this study highlights the reciprocal relation between amygdalar and ventromedial prefrontal cortex in the encoding of emotional valence as reflected by facial expression.